AP-endonuclease (APE) plays a key role in repairing oxidative DNA damage generated endogenously or induced by reactive oxygen species (ROS) resulting from ionizing radiation and other agents. APE acts as an abasic site endonuclease, and as a DNA 3' phosphoesterase to remove 3'- blocking groups generated directly or indirectly by ROS. The latter activity may be limiting in human and mouse cells and thus the cellular level of APE may determine the repair efficiency of ROS-induced DNA strand breaks. The 36 kDa major human APE (hAPE1) (also identified as Ref1), is a multifunctional protein. It acts as a reductive activator of transcription factors, (e.g., c-Jun and p53), and also as a (co)repressor of the parathyroid hormone and possibly other genes by binding to two types of negative Ca2+-response element (nCaRE-A and B). APE1 regulation is complex. In addition to its transient activation by ROS, the protein is downregulated during apoptosis and differentiation in some cells. The hAPE1 gene may also be autoregulated because of the presence of nCaRE's in its own promoter. A second protein (hnRNP-L or Ku86) is needed for binding to nCaRE-B and nCaRE-A respectively. Not surprisingly, early embryonic lethality was observed in APE1 nullizygous mice, and no APE 1-cell line has yet been established. The broad objective of this project is to elucidate the molecular bases for APE1 regulation. Furthermore, identifying its essential in vivo role may show the linkage among its apparently unrelated functions. The specific aims of this competing renewal application are: (1) To test the hypothesis that APE1 forms a functional repressor in vitro by complexing with hnRNP-L and/or Ku86 in order to bind to nCaREs. (2) To test the hypothesis that the APE1 gene is autoregulated in the mouse, by using transgenic mice with distinct numbers of integrated hAPE1 gene copies which have already been generated. (3) To elucidate the signaling processes in APE1 regulation, including characterization of APE1 phosphorylation in vivo and to identify and characterize unknown cis elements in the hAPE1 promoter. (4) To test the hypothesis that the requirement for hAPE1 in the mouse embryo is due to its role in transcriptional regulation, by generating and characterizing conditional knockout mouse mutants and cells derived therefrom. The long-term goal of this project is a comprehensive understanding of the interlocking regulatory circuits involving APE 1 in cellular homeostasis, which have a direct impact on the cellular oxidative damage response and DNA repair.